Methionine is one of essential amino acids of human body which has been widely used as feed and food additives and further used as a synthetic raw material for medical solutions and medical supplies. Methionine acts as a precursor of such compounds as choline (lecithin) and creatine and at the same time is used as a synthetic raw material for cysteine and taurine. Methionine can also provide sulfur. S-adenosyl-methionine is derived from L-methionine and plays a certain role in providing methyl group in human body and also is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) inhibits fat accumulation in the liver and artery and alleviates depression, inflammation, liver disease, and muscle pain, etc.
The in vivo functions of methionine and/or S-adenosyl-L-methionine known so far are as follows.
1) It inhibits fat accumulation in the liver and artery promoting lipid metabolism and improves blood circulation in the brain, heart and kidney (J. Hepatol. Jeon B R et al., 2001 Mar; 34(3): 395-401).
2) It promotes digestion, detoxication and excretion of toxic substances and excretion of heavy metals such as Pb.
3) It can be administered as an anti-depression agent at the dosage of 800-1,600 mg/day (Am J Clin Nutr. Mischoulon D. et al., 2002 Nov; 76(5): 1158S-61S).
4) It enhances liver functions (FASEB J. Mato J M. 2002 Jan; 16(1): 15-26) and particularly is effective in the liver disease caused by alcohol (Cochrane Database Syst Rev., Rambaldi A., 2001; (4): CD002235).
5) It has anti-inflammatory effect on bone and joint diseases and promotes joint-recovery (ACP J Club. Sander O., 2003 Jan-Feb; 138(1): 21, J Fam Pract., Soeken K L et al., 2002 May; 51(5): 425-30).
6) It is an essential nutrient for hair. It provides nutrition to hair and thereby prevents hair loss (Audiol Neurootol., Lockwood D S et al., 2000 Sep-Oct; 5(5): 263-266).
Methionine can be chemically or biologically synthesized to be applied to feed, food and medicines,
In the chemical synthesis, methionine is mostly produced by hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. The chemically synthesized methionine has a disadvantage of only being produced as a mixed form of L-type and D-type.
In the biological systhesis, methionine is produced by method the using proteins involved in methionine synthesis. L-methionine is biosynthesized from homoserine by the action of the enzyme expressed by such genes as metA, metB, metC, metE and metH in Escherichia coli. Particularly, metA is the gene encoding homoserine O-succinyltransferase which is the first enzyme necessary for methionine biosynthesis, and it converts homoserine into O-succinyl-L-homoserine. O-succinylhomoserine lyase or cystathionine gamma synthase encoded by metB gene converts O-succinyl-L-homoserine into cystathionine. Cystathionine beta lyase encoded by metC gene converts cystathionine into L-homocysteine. MetE encodes cobalamine-independent methionine synthase and metH encodes cobalamine-dependent methionine synthase, both of which convert L-homocysteine into L-methionine. At this time, 5,10-methylenetetrahydrofolate reductase encoded by metF and serine hydroxymethytransferase encoded by glyA work together to synthesize N(5)-methyltetrahydrofolate providing methyl group necessary for L-methionine synthesis.
L-methionine is synthesized by a series of organic reactions by the above enzymes. The genetic modification on the above proteins or other proteins affecting the above proteins might result in the increase of L-methionine synthesis. For example, Japanese Laid-Open Patent Publication No. 2000/139471 describes a method of producing L-methionine with the Escherichia sp. of which thrBC and met.) genes on the genome are deleted, metBL is over-expressed and metK is replaced by a leaky mutant. Also, US Patent Publication No. US2003/0092026 A1 describes a method using a metD (L-methionine synthesis inhibitor) knock-out microorganism which belongs to Corynerbacterium sp. US Patent Publication No. US2002/0049305 describes a method to increase L-methionine production by increasing the expression of 5,10-methylenetetrahydrofolate reductase (metF).
The methionine produced by the biological method is L-type, which has advantages but the production amount is too small. This is because the methionine biosynthetic pathway has very tight feed-back regulation systems. Once methionine is synthesized to a certain level, the final product methionine inhibits the transcription of metA gene encoding the primary protein for initiation of methionine biosynthesis. Over-expression of metA gene itself cannot increase methionine production because the metA gene is suppressed by methionine in the transcription stage and then degraded by the intracellular proteases in the translation stage (Dvora Biran, Eyal Gur, Leora Gollan and Eliora Z. Ron: Control of methionine biosynthesis in Escherichia coli by proteolysis: Molecular Microbiology (2000) 37(6), 1436-1443). Therefore, many of previous patents were focused on how to free the metA gene from its feed-back regulation system (WO2005/108561, WO1403813).
US patent publication No. US2005/0054060A1 describes a method to synthesize homocysteine or methionine by modified cystathionine synthase (O-succinylhomoserine lyase) which use methylmercaptan (CH3SH) or hydrogen sulfide (H2S) directly as a sulfur source, not cysteine. However, it is well understood by those in the art that cystathionine synthase can bind various methionine precursor in the cells and thereby produce by-product al high level. For example, it was reported that cystathionine synthase accumulate high levels of homolanthionin by side reaction of O-succinylhomoserine and homocystein (J. Bacteriol (2006) vol 188:p609-618). Therefore, overexpression of cystathionine synthase can reduce the efficiency Intracellular reaction due to the increase of their side reaction. In addition, this method has many disadvantages. This process uses intracellular metabolic pathways which have side reactions and feed back regulation systems. Also this process uses H2S or CH3SH which has a severe cytotoxity to cells. Hence the methionine production yield is comparatively small.
To solve these problems, the present inventor had developed two step process comprising; first step of producing of L-methionine precursor by E.coli fermentation; and second step of converting L-methionine precursor into L-methionine by enzyme reaction (PCT/KR2007/003650), the contents of which is incorporated herein by reference. This two step process can solve above problems, such as cytotoxicity of sulfides, feed-back regulation by methionine and SAMe, and decomposition intermediate product by intracellular enzymes (e.g. cystathionine gamma synthase, O-succinylhoinoserine sulfhydrylase and O-acetylhoraoserine sulfydrylase). Morever, against the chemical methionine synthetic method which produce mixed form of D-methionine and L-methionine, the two step process is very efficient to produce only L-methionine selectively.
In this two step process, production yield of methionine precursor is one of the key factor for the increase of methionine procution yield. To increase the synthetic yield of methionine precursor, O-succinyl homoserine, good combination of strong aspartokinase, homoserine transferase and feed back resistant O-succinyl homoserine transferase is really important. On the above-mentioned background, the present inventors were produced a microorganism which can produce O-succinylhomoserine characterized by the followings: 1) the homoserine O-succinyltransferase activity is introduced and enhanced, wherein the homoserine O-succinyltransferase activity is feed back resistant to methionine; and 2) the aspartokinase or homoserine dehydrogenase activity (EC2.7.2.4 or 1.1.1.3) is enhanced. This strain is capable of producing high concentrations of L-methionine precursor regardless of methionine concentration in the culture medium and production of L-methionine precursor can be remarkably increased.